Heterobifunctional pan-selectin inhibitors

ABSTRACT

Compounds and methods are provided for modulating in vitro and in vivo processes mediated by selectin binding. More specifically, selectin modulators and their use are described, wherein the selectin modulators that modulate (e.g., inhibit or enhance) a selectin-mediated function comprise particular glycomimetics alone or linked to a member of a class of compounds termed BASAs (Benzyl Amino Sulfonic Acids) or a member of a class of compounds termed BACAs (Benzyl Amino Carboxylic Acids).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/515,343 filed Sep. 1, 2006, now allowed; which application claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationNo. 60/713,994 filed Sep. 2, 2005, which applications are incorporatedherein by reference in their entirety.

BACKGROUND

1. Technical Field

The present invention relates generally to compounds, compositions andmethods for modulating processes mediated by selectin binding, and moreparticularly to selectin modulators and their use, wherein the selectinmodulators that modulate a selectin-mediated function compriseparticular glycomimetics alone or linked to a member of a class ofcompounds termed BASAs (Benzyl Amino Sulfonic Acids) or a member of aclass of compounds termed BACAs (Benzyl Amino Carboxylic Acids).

2. Description of the Related Art

When a tissue is infected or damaged, the inflammatory process directsleukocytes and other immune system components to the site of infectionor injury. Within this process, leukocytes play an important role in theengulfment and digestion of microorganisms. Thus, the recruitment ofleukocytes to infected or damaged tissue is critical for mounting aneffective immune defense.

Selectins are a group of structurally similar cell surface receptorsthat are important for mediating leukocyte binding to endothelial cells.These proteins are type 1 membrane proteins and are composed of an aminoterminal lectin domain, an epidermal growth factor (EGF)-like domain, avariable number of complement receptor related repeats, a hydrophobicdomain spanning region and a cytoplasmic domain. The bindinginteractions appear to be mediated by contact of the lectin domain ofthe selectins and various carbohydrate ligands.

There are three known selectins: E-selectin, P-selectin and L-selectin.E-selectin is found on the surface of activated endothelial cells, whichline the interior wall of capillaries. E-selectin binds to thecarbohydrate sialyl-Lewis^(x) (SLe^(x)), which is presented as aglycoprotein or glycolipid on the surface of certain leukocytes(monocytes and neutrophils) and helps these cells adhere to capillarywalls in areas where surrounding tissue is infected or damaged; andE-selectin also binds to sialyl-Lewis^(a) (SLe^(a)), which is expressedon many tumor cells. P-selectin is expressed on inflamed endothelium andplatelets, and also recognizes SLe^(x) and SLe^(a), but also contains asecond site that interacts with sulfated tyrosine. The expression ofE-selectin and P-selectin is generally increased when the tissueadjacent to a capillary is infected or damaged. L-selectin is expressedon leukocytes. Selectin-mediated intercellular adhesion is an example ofa selectin-mediated function.

Modulators of selectin-mediated function include the PSGL-1 protein (andsmaller peptide fragments), fucoidan, glycyrrhizin (and derivatives),anti-selectin antibodies, sulfated lactose derivatives, and heparin. Allhave shown to be unsuitable for drug development due to insufficientactivity, toxicity, lack of specificity, poor ADME characteristicsand/or availability of material.

Although selectin-mediated cell adhesion is required for fightinginfection and destroying foreign material, there are situations in whichsuch cell adhesion is undesirable or excessive, resulting in tissuedamage instead of repair. For example, many pathologies (such asautoimmune and inflammatory diseases, shock and reperfusion injuries)involve abnormal adhesion of white blood cells. Such abnormal celladhesion may also play a role in transplant and graft rejection. Inaddition, some circulating cancer cells appear to take advantage of theinflammatory mechanism to bind to activated endothelium. In suchcircumstances, modulation of selectin-mediated intercellular adhesionmay be desirable.

Accordingly, there is a need in the art for identifying inhibitors ofselectin-mediated function, e.g., of selectin-dependent cell adhesion,and for the development of methods employing such compounds to inhibitconditions associated with excessive selectin activity. The presentinvention fulfills these needs and further provides other relatedadvantages.

BRIEF SUMMARY

Briefly stated, this invention provides compounds, compositions andmethods for modulating selectin-mediated processes. In the presentinvention, the compounds that modulate (e.g., inhibit or enhance) aselectin-mediated function comprise a particular glycomimetic alone orlinked to a BASA or a BACA. Such compounds may be combined with apharmaceutically acceptable carrier or diluent to form a pharmaceuticalcomposition. The compounds or compositions may be used in a method tomodulate (e.g., inhibit or enhance) a selectin-mediated function, suchas inhibiting a selectin-mediated intercellular adhesion.

In one aspect of the present invention, compounds are provided havingthe formula:

wherein:

where n=0-2, and R⁸ are independently selected where n=2;

-   -   R²═H, —C(═O)OX where X is C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈        alkynyl or C₁-C₁₄ aryl, —(═O)NH(CH₂)_(n)NH₂,        -   —[C(═O)NH(CH₂)_(n)NHC(═O)]_(m)(L)_(m)Z, where n=0-30, m=0-1,            L is a linker, and Z is a benzyl amino sulfonic acid, a            benzyl amino carboxylic acid, a polyethylene glycol, or a            second compound or salt thereof having the above formula to            form a dimer where R² of the second compound or salt thereof            has m=0, no Z, and is the point of attachment;    -   R³═—OH,

-   -   -   —O—C(═O)—X, —NH₂, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2            and X is independently selected from C₁-C₈ alkanyl, C₁-C₈            alkenyl, C₁-C₈ alkynyl,

-   -   -   and any of the above ring compounds may be substituted with            one to three independently selected of Cl, F, C₁-C₈ alkanyl,            C₁-C₈ alkenyl, C₁-C₈ alkynyl, C₁-C₁₄ aryl, or OY where Y is            H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, or C₁-C₁₄            aryl;

-   -   -   6′sulfated GlcNAc, 6′carboxylated GlcNAc, 6′sulfated GalNAc,            6′sulfated galactose, 6′carboxylated galactose or

-   -   -   where R⁹ is aryl, heteroaryl, cyclohexane, t-butane,            adamantane, or triazole, and any of R⁹ may be substituted            with one to three independently selected of Cl, F, C₁-C₈            alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or OY where Y is H,            C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or C₁-C₁₄ aryl;

    -   R⁵═H, or R⁴ and R⁵ are taken together to form

-   -   -   where R¹⁰ is aryl, heteroaryl,

-   -   -   where n=0-10, and any one of the above ring compounds may be            substituted with one to three independently selected of Cl,            F, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or OY where Y            is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl or C₁-C₈ alkynyl;

    -   R⁶═H, fucose, mannose, arabinose, galactose or polyols;

    -   R⁷═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or

-   -   R⁸═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,

-   -   -   where n=0-3 and X is independently selected from H, OH, Cl,            F, N₃, NH₂, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,            C₁-C₁₄ aryl, OC₁-C₈ alkanyl, OC₁-C₈ alkenyl, OC₁-C₈ alkynyl,            and OC₁-C₁₄ aryl, and any of the above ring compounds may be            substituted with one to three independently selected of Cl,            F, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, C₁-C₁₄ aryl            or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈            alkynyl, or C₁-C₁₄ aryl.

A compound of the present invention includes physiologically acceptablesalts thereof. A compound of the present invention in combination with apharmaceutically acceptable carrier or diluent provides a composition ofthe present invention. In the chemical formulae herein, a line extendingfrom an atom depicted or a carbon implied by the intersection of the twoother lines, represents the point of attachment (and does not representa methyl group).

In an embodiment of the present invention, R⁶ is fucose:

In an embodiment, R⁷ is H.

In an embodiment, R⁴ is

In an embodiment, R⁴ is

where R⁹ is defined as for the general formula above.

In an embodiment, R⁹ is cyclohexane.

In an embodiment, R⁶ is galactose.

In an embodiment, R⁸ is

In an embodiment, R² is —[C(═O)NH(CH₂)_(n)NHC(═O)]_(m)(L)_(m)Z, where n,m, L and Z are defined as for the general formula above.

In an embodiment, Z is a benzyl amino sulfonic acid, a benzyl aminocarboxylic acid or a polyethylene glycol.

In an embodiment, R³ is —O—C(═O)—X, where X is defined as for thegeneral formula above.

In an embodiment, X is

In an embodiment, R⁵ is H.

In an embodiment, L is a polyethylene glycol or a thiadiazole.

In an embodiment, a compound comprises a compound according to thepresent invention, further comprising a diagnostic or therapeutic agent.Such a compound may be combined with a pharmaceutically acceptablecarrier or diluent to form one embodiment of a composition of thepresent invention.

In another aspect of the present invention, methods are provided forusing a compound or composition of the present invention to modulate aselectin-mediated function. Such a compound or composition can be used,for example, to inhibit or enhance a selectin-mediated function, such asselectin-mediated intercellular interactions. A compound or compositioncan be used in a method to contact a cell expressing a selectin in anamount effective to modulate the selectin's function. A compound orcomposition can be used in a method to administer to a patient, who isin need of having inhibited the development of a condition associatedwith an excessive selectin-mediated function (such as an excessiveselectin-mediated intercellular adhesion), in an amount effective toinhibit the development of such a condition. Examples of such conditionsinclude inflammatory diseases, autoimmune diseases, infection, cancer,shock, thrombosis, wounds, burns, reperfusion injury, platelet-mediateddiseases, leukocyte-mediated lung injury, spinal cord damage, digestivetract mucous membrane disorders, osteoporosis, arthritis, asthma andallergic reactions. A compound or composition can be used in a method toadminister to a patient who is the recipient of a transplanted tissue inan amount effective to inhibit rejection of the transplanted tissue. Acompound or composition can be used in a method in an amount effectiveto target an agent (e.g., a diagnostic or therapeutic agent) to aselectin-expressing cell by contacting such a cell with the agent linkedto the compound or composition. A compound or composition can be used inthe manufacture of a medicament, for example for any of the uses recitedabove.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are diagrams illustrating the syntheses of BASAs.

FIG. 2 is a diagram illustrating the synthesis of a BACA.

FIG. 3 is a diagram illustrating the synthesis of a glycomimetic (XIX).

FIG. 4 is a diagram illustrating the synthesis of a glycomimetic(XXVIII).

FIG. 5 is a diagram illustrating the synthesis of a PEGylatedglycomimetic (XXX).

FIG. 6 is a diagram illustrating the synthesis of a PEGylatedglycomimetic (XXXI).

FIGS. 7A, 7B, and 7C are diagrams illustrating the syntheses ofPEGylated BASAs (XXXII and XXXIII) and PEGylated BACAs (XXXVI andXXXVIa).

FIGS. 8A, 8B and 8C are diagrams illustrating the syntheses ofGlycomimetic-BASA (FIGS. 8A and 8C) and Glycomimetic-BACA (FIG. 8B).

FIGS. 9A, 9B and 9C are diagrams illustrating the syntheses ofGlycomimetic-BASA (FIGS. 9A and 9C) and Glycomimetic-BACA (FIG. 9B).

FIG. 10 is a diagram illustrating the synthesis of a glycomimetic.

FIG. 11 is a diagram illustrating the synthesis of a glycomimetic.

FIG. 12 is a diagram illustrating the synthesis of a glycomimetic.

FIG. 13 is a diagram illustrating the synthesis of a glycomimetic-BASA.

FIG. 14 is a diagram illustrating the synthesis of a glycomimetic-BASA.

FIG. 15 is a diagram illustrating the synthesis of a glycomimetic-BASA.

FIG. 16 is a diagram illustrating the synthesis of glycomimetics.

FIGS. 17A and 17B show a comparison of the activity of glycomimeticinhibitors in binding assays for E-selectins (FIG. 17A) and P-selectins(FIG. 17B) in vitro. A and B are glycomimetic-BASAs other than thepresent invention. C is the glycomimetic-BASA of FIG. 8C.

FIG. 18 shows the effect of glycomimetic-BASA of FIG. 8C on neutrophilmigration. A is a vehicle only and E is a positive control (mixedantibodies). B, C and D are the glycomimetic-BASA of FIG. 8C at a doseof 5 mg/kg, 10 mg/kg, and 20 mg/kg, respectively.

FIG. 19 shows a comparison of the effects of glycomimetic inhibitors onneutrophil migration in murine air pouch model. A is IL-1β+vehicle. B isIL-1β+the glycomimetic-BASA of FIG. 13. C is IL-1β+the glycomimetic-BASAof FIG. 8C. D is IL-1β+mixed antibodies (positive control). *P<0.05 vs.A (vehicle group).

DETAILED DESCRIPTION

As noted above, the present invention provides selectin modulators,compositions thereof and methods for modulating selectin-mediatedfunctions. Such modulators may be used in vitro or in vivo, to modulate(e.g., inhibit or enhance) selectin-mediated functions in a variety ofcontexts, discussed in further detail below. Examples ofselectin-mediated functions include intercellular adhesion and theformation of new capillaries during angiogenesis.

Selectin Modulators

The term “selectin modulator,” as used herein, refers to a molecule(s)that modulates (e.g., inhibits or enhances) a selectin-mediatedfunction, such as selectin-mediated intercellular interactions. Aselectin modulator may consist entirely of a glycomimetic compound ofthe present invention, or may consist of such a glycomimetic linked to aBASA (Benzyl Amino Sulfonic Acid) or a BACA (Benzyl Amino CarboxylicAcid), or may comprise one or more additional molecular components toany of the above.

A selectin modulator of the present invention which does not possess aBASA or a BACA is preferably used to inhibit an E-selectin-mediatedfunction. With the addition of a BASA or BACA to a glycomimetic of thepresent invention, the selectin modulator has increased ability tomodulate P- and L-selectin-mediated functions as well.

A selectin modulator of the present invention is a compound orphysiologically acceptable salt thereof, having the formula:

wherein:

where n=0-2, and R⁸ are independently selected where n=2;

-   -   R²═H, —C(═O)OX where X is C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈        alkynyl or C₁-C₁₄ aryl, —C(═O)NH(CH₂)_(n)NH₂,        -   —[C(═O)NH(CH₂)_(n)NHC(═O)]_(m)(L)_(m)Z, where n=0-30, m=0-1,            L is a linker, and Z is a benzyl amino sulfonic acid, a            benzyl amino carboxylic acid, a polyethylene glycol, or a            second compound or salt thereof having the above formula to            form a dimer where R² of the second compound or salt thereof            has m=0, no Z, and is the point of attachment;    -   R³═—OH,

-   -   -   —O—C(═O)—X, —NH₂, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2            and X is independently selected from C₁-C₈ alkanyl, C₁-C₈            alkenyl, C₁-C₈ alkynyl,

-   -   -   and any of the above ring compounds may be substituted with            one to three independently selected of Cl, F, C₁-C₈ alkanyl,            C₁-C₈ alkenyl, C₁-C₈ alkynyl, C₁-C₁₄ aryl, or OY where Y is            H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, or C₁-C₁₄            aryl;

-   -   -   6′sulfated GlcNAc, 6′carboxylated GlcNAc, 6′sulfated GalNAc,            6′sulfated galactose, 6′carboxylated galactose or

-   -   -   where R⁹ is aryl, heteroaryl, cyclohexane, t-butane,            adamantane, or triazole, and any of R⁹ may be substituted            with one to three independently selected of Cl, F, C₁-C₈            alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or OY where Y is H,            C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or C₁-C₁₄ aryl;

    -   R⁵═H, or R⁴ and R⁵ are taken together to form

-   -   -   where R¹⁹ is aryl, heteroaryl,

-   -   -   where n=0-10, and any one of the above ring compounds may be            substituted with one to three independently selected of Cl,            F, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or OY where Y            is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl or C₁-C₈ alkynyl;

    -   R⁶═H, fucose, mannose, arabinose, galactose or polyols;

    -   R⁷═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or

-   -   R⁸═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,

-   -   -   where n=0-3 and X is independently selected from H, OH, Cl,            F, N₃, NH₂, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,            C₁-C₁₄ aryl, OC₁-C₈ alkanyl, OC₁-C₈ alkenyl, OC₁-C₈ alkynyl,            and OC₁-C₁₄ aryl, and any of the above ring compounds may be            substituted with one to three independently selected of Cl,            F, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, C₁-C₁₄ aryl            or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈            alkynyl, or C₁-C₁₄ aryl.

As used herein, a “C₁-C₈ alkanyl” refers to an alkane substituent withone to eight carbon atoms and may be straight chain or branched.Examples are methyl, ethyl, propyl, isopropyl, butyl and t-butyl. A“C₁-C₈ alkenyl” refers to an alkene substituent with one to eight carbonatoms, at least one carbon-carbon double bond, and may be straight chainor branched. Examples are similar to “C₁-C₈ alkanyl” examples exceptpossessing at least one carbon-carbon double bond. A “C₁-C₈ alkynyl”refers to an alkyne substituent with one to eight carbon atoms, at leastone carbon-carbon triple bond, and may be straight chain or branched.Examples are similar to “C₁-C₈ alkanyl” examples except possessing atleast one carbon-carbon triple bond. An “aryl” refers to an aromaticsubstituent with one to fourteen carbon atoms in one or multiple ringswhich may be separated by a bond or fused. A “heteroaryl” is similar toan “aryl” except the aromatic substituent possesses at least oneheteroatom (such as N, O or S) in place of a ring carbon. Examples ofaryls and heteroaryls include phenyl, naphthyl, pyridinyl, pyrimidinyl,triazolo, furanyl, oxazolyl, thiophenyl, quinolinyl and diphenyl. Asused herein, the term “independently selected” refers to the selectionof identical or different substituents.

As used herein, polyethylene glycol (“PEG”) refers to multiple units ofethylene glycol, as well as those with one or more substituents (e.g.,dicarboxylated PEG). PEGs with and without substituents are well knownto those in the art. Within the present invention, PEG can serve as asubstituent on a selectin modulator, or as a linker to attach othergroups or compounds to a selectin modulator, or a selectin modulator maypossess more than one PEG.

Where a second selectin modulator is linked to a first selectinmodulator, a dimer of selectin modulators (i.e., a divalent molecule) isformed. A variety of linkers may be used to join the two selectinmodulators. For example, PEG may be used as the linker to prepare adimer. As used herein, a “dimer” can be a homodimer or a heterodimer. Ahomodimer refers to a dimer where the two selectin modulators joinedtogether are identical (independent of the substituents for the linkingto one another). A heterodimer refers to a dimer where the two selectinmodulators (independent of the linkage substituents) are not identical.

A selectin modulator of the present invention may possess, at R⁴ of theabove formula, sialic acid or a sialic acid mimic as set forth above.For example, the hexose ring of sialic acid may be replaced withcyclohexane. The presence of sialic acid in the selectin modulatorenhanced P-selectin binding. Where only E-selectin binding (and not bothE- and P-selecting binding) is desired, a sialic acid mimic replacessialic acid in the selectin modulator.

Alternative to (or in combination with) the replacement of a sialic acidmimic with sialic acid, P-selectin binding may be enhanced by theaddition of a BASA or a BACA. As disclosed above, the selectin modulatorcompounds of the present invention may possess a “Z” at R², and Z may bea BASA or a BACA. The addition of a BASA or BACA to a selectin modulatorcompound of the present invention that lacks sialic acid, can convertthe selectin modulator from a compound that is selective for binding toE-selectin to one that binds both E- and P-selectin. BASA or BACAincludes a portion or an analogue of a BASA or BACA or portion ofeither, provided that the compound retains the ability to modulate aselectin-mediated function. PEG may be added to a selectin modulatorwith or without a BASA (or BACA). PEG may also be used to link a BASA orBACA to a selectin modulator.

Within the present invention, BASAs are low molecular weight sulfatedcompounds which have the ability to interact with a selectin. Theinteraction modulates or assists in the modulation (e.g., inhibition orenhancement) of a selectin-mediated function (e.g., an intercellularinteraction). They exist as either their protonated acid form, or as asodium salt, although sodium may be replaced with potassium or any otherpharmaceutically acceptable counterion. A representative BASA has thefollowing structure:

Portions of BASA that retain the ability to interact with a selectin(which interaction modulates or assists in the modulation of aselectin-mediated function as described herein) are also a BASAcomponent of the selectin modulators of the present invention. Suchportions generally comprise at least one aromatic ring present withinthe BASA structure. Within certain embodiments, a portion may comprise asingle aromatic ring, multiple such rings or half of a symmetrical BASAmolecule.

As noted above, analogues of BASA and portions thereof (both of whichpossess the biological characteristic set forth above) are alsoencompassed, e.g., by the BASA component of the selectin modulators,within the present invention. As used herein, an “analogue” is acompound that differs from BASA or a portion thereof because of one ormore additions, deletions and/or substitutions of chemical moieties,such that the ability of the analogue to inhibit a selectin-mediatedinteraction is not diminished. For example, an analogue may contain S toP substitutions (e.g., a sulfate group replaced with a phosphate group).Other possible modifications include: (a) modifications to ring size(e.g., any ring may contain between 4 and 7 carbon atoms); (b)variations in the number of fused rings (e.g., a single ring may bereplaced with a polycyclic moiety containing up to three fused rings, apolycyclic moiety may be replaced with a single unfused ring or thenumber of fused rings within a polycyclic moiety may be altered); (c)ring substitutions in which hydrogen atoms or other moieties covalentlybonded to a carbon atom within an aromatic ring may be replaced with anyof a variety of moieties, such as F, Cl, Br, I, OH, O-alkyl (C1-8), SH,NO₂, CN, NH₂, NH-alkyl (C1-8), N-(alkyl)₂, SO₃M (where M=H⁺, Na⁺, K⁺ orother pharmaceutically acceptable counterion), CO₂M, PO₄M₂, SO₂NH₂,alkyl (C1-8), aryl (C6-10), CO₂-alkyl (C1-8), —CF₂X (where X can be H,F, alkyl, aryl or acyl groups) and carbohydrates; and (d) modificationsto linking moieties (i.e., moieties located between rings in the BASAmolecule) in which groups such as alkyl, ester, amide, anhydride andcarbamate groups may be substituted for one another.

Certain BASA portions and analogues contain one of the following genericstructures:

Within this structure, n may be 0 or 1, X¹ may be —PO₂M, —SO₂M or —CF₂—(where M is a pharmaceutically acceptable counterion such as hydrogen,sodium or potassium), R¹ may be —OH, —F or —CO₂R⁴ (where R⁴ may be —H or—(CH₂)_(m)—CH₃ and m is a number ranging from 0 to 3, R² may be —H,—PO₃M₂, —SO₃M₂, —CH₂—PO₃M₂, —CH₂—SO₃M₂, —CF₃ or —(CH₂)_(m)—C(R⁶)H—R⁵ orR⁹—N(R¹⁰)—, R³ may be —H, —(CH₂)_(m)—C(R⁶)H—R⁵ or R⁹—N(R¹⁰)— (where R⁵and R⁶ may be independently selected from —H, —CO₂—R⁷ and —NH—R⁸, R⁷ andR⁸ may be independently selected from hydrogen and moieties comprisingone or more of an alkyl group, an aromatic moiety, an amino group or acarboxy group, and R⁹ and R¹⁹ may be independently selected from —H,—(CH₂)_(m)—CH₃; —CH₂—Ar, —CO—Ar, where m is a number ranging from 0 to 3and Ar is an aromatic moiety (i.e., any moiety that comprises at leastone substituted or unsubstituted aromatic ring, wherein the ring isdirectly bonded to the —CH₂— or —CO— group indicated above)).

Other portions and analogues of BASA comprise the generic structure:

Within this structure, R₁ and R₂ may be independently selected from (i)hydrogen, (ii) moieties comprising one or more of an alkyl group, anaromatic moiety, an amino group or a carboxy group, and (iii) —CO—R₃(where R₃ comprises an alkyl or aromatic moiety as described above) andM is a pharmaceutically acceptable counterion.

The individual compounds, or groups of compounds, derived from thevarious combinations of the structures and substituents describedherein, are disclosed by the present application to the same extent asif each compound or group of compounds was set forth individually. Thus,selection of particular structures and/or particular substituents iswithin the scope of the present invention.

Representative BASA portions and analogues are included in the compoundsshown in FIGS. 1A-1B. It will be apparent to those of ordinary skill inthe art that modifications may be made to the compounds shown withinthese figures, without adversely affecting the ability to function asselectin modulators. Such modifications include deletions, additions andsubstitutions as described above.

A BACA is similar to a BASA, except instead of sulfonic acid groups, thecompound possesses carboxylic acid groups. For example, the sulfonicacid groups of the above BASA compounds may be replaced with carboxylicacid groups. Thus, the above disclosure to BACAs is incorporated byreference into this description of BACAs.

Examples of BACAs include:

where X is F or Cl; Y is H, —C(═O)(O—CH₂CH₂)_(n) or —C(═O)(CH₂)_(n)wherein n=0-8; and Z is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl or C₁-C₈alkynyl.

As described above, a BASA or BACA may be joined to a compound of thepresent invention at R² via a linker (“L”). Typically a linker is firstattached to one of a glycomimetic or a BASA/BACA, which is then reactedwith the other. The attachment of a BASA or BACA to a particularglycomimetic can be accomplished in a variety of ways to form a selectinmodulator. A linker possessed by (or added to) a BASA or BACA or aglycomimetic may include a spacer group, such as —(CH₂)_(n)— or—O(CH₂)_(n)— where n is generally about 1-20 (including any wholeinteger range therein). An example of a linker is —NH₂ on aglycomimetic, e.g., —CH₂—NH₂ when it includes a short spacer group. Inan embodiment, —CH₂—NH₂ is attached to a glycomimetic at R′ which maythen be used to attach a BASA or BACA. The simplest attachment method isreductive amination of the BASA or BACA to a glycomimetic containing areducing end (an anomeric hydroxyl/aldehyde). This is accomplished bysimple reaction of the BASA or BACA to the reducing end and subsequentreduction (e.g., with NaCNBH₃ at pH 4.0) of the imine formed. The mostgeneral approach entails the simple attachment of an activated linker tothe glycomimetic via an O, S or N heteroatom (or C atom) at the anomericposition. The methodology of such attachments has been extensivelyresearched for carbohydrates and anomeric selectivity is easilyaccomplished by proper selection of methodology and/or protectinggroups. Examples of potential glycosidic synthetic methods include Lewisacid catalyzed bond formation with halogen or peracetylated sugars(Koenigs Knorr), trichloroacetamidate bond formation, thioglycosideactivation and coupling, glucal activation and coupling, n-pentenylcoupling, phosphonate ester homologation (Horner-Wadsworth-Emmonsreaction), and many others. Alternatively, linkers could be attached topositions on the moieties other than the anomeric. The most accessiblesite for attachment is at a six hydroxyl (6-OH) position of aglycomimetic (a primary alcohol). The attachment of a linker at the 6-OHcan be easily achieved by a variety of means. Examples include reactionof the oxy-anion (alcohol anion formed by deprotonation with base) withan appropriate electrophile such as an alkyl/acyl bromide, chloride orsulfonate ester, activation of the alcohol via reaction with a sulfonateester chloride or POCl₃ and displacement with a subsequent nucleophile,oxidation of the alcohol to the aldehyde or carboxylic acid forcoupling, or even use of the Mitsunobu reaction to introduce differingfunctionalities. Once attached the linker is then functionalized forreaction with a suitable nucleophile on the BASA or BACA (or viceversa). This is often accomplished by use of thiophosgene and amines tomake thiourea-linked heterobifunctional ligands, diethyl squarateattachment (again with amines) and/or simple alkyl/acylation reactions.Additional methods that could be utilized include FMOC solid or solutionphase synthetic techniques traditionally used for carbohydrate andpeptide coupling and chemo-enzymatic synthesis techniques possiblyutilizing glycosyl/fucosyl transferases and/or oligosaccharyltransferase(OST).

Embodiments of linkers include the following:

Other linkers (e.g., PEG) will be familiar to those in the art or inpossession of the present disclosure.

A compound, or physiologically acceptable salt thereof, of the presentinvention has the formula:

wherein R¹-R⁹ are defined as set forth above.

In a preferred embodiment, R⁶ is fucose:

In a preferred embodiment, R⁷ is H. In a preferred embodiment, R⁴ is

In a preferred embodiment, R⁴ is

where R⁹ is defined as above. In a preferred embodiment, R⁹ iscyclohexane. In a preferred embodiment. R⁶ is galactose. In a preferredembodiment, R⁸ is

In a preferred embodiment, R² is—[C(═O)NH(CH₂)_(n)NHC(═O)]_(m)(L)_(m)Z, where n, m, L and Z are definedas above. In a preferred embodiment, Z is a benzyl amino sulfonic acid,a benzyl amino carboxylic acid or a polyethylene glycol. In a preferredembodiment, R³ is —O—C(═O)—X, where X is defined as above. In apreferred embodiment, X is

In a preferred embodiment, R⁵ is H. In a preferred embodiment, L is apolyethylene glycol or a thiadiazole.

Although selectin modulators as described herein may sufficiently targeta desired site in vivo, it may be beneficial for certain applications toinclude an additional targeting moiety to facilitate targeting to one ormore specific tissues. As used herein, a “targeting moiety,” may be anysubstance (such as a compound or cell) that, when linked to a modulatingagent enhances the transport of the modulator to a target tissue,thereby increasing the local concentration of the modulator. Targetingmoieties include antibodies or fragments thereof, receptors, ligands andother molecules that bind to cells of, or in the vicinity of, the targettissue. Linkage is generally covalent and may be achieved by, forexample, direct condensation or other reactions, or by way of bi- ormulti-functional linkers.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a selectin modulator. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds.Examples of potential drugs include antineoplastic agents (such as5-fluorouracil and distamycin), integrin agonist/antagonists (such ascyclic-RGD peptide), cytokine agonist/antagonists, histamineagonist/antagonists (such as diphenhydramine and chlorpheniramine),antibiotics (such as aminoglycosides and cephalosporins) and redoxactive biological agents (such as glutathione and thioredoxin). In otherembodiments, diagnostic or therapeutic radionuclides may be linked to aselectin modulator. In many embodiments, the agent may be linkeddirectly or indirectly to a selectin modulator.

Modulators as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or moremodulators in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Within yet other embodiments, compositions of the present invention maybe formulated as a lyophilizate. Compositions of the present inventionmay be formulated for any appropriate manner of administration,including for example, topical, oral, nasal, intravenous, intracranial,intraperitoneal, subcutaneous, or intramuscular administration.

A pharmaceutical composition may also, or alternatively, contain one ormore active agents, such as drugs (e.g., those set forth above), whichmay be linked to a modulator or may be free within the composition.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of modulating agent followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Carriers for use within such formulations are biocompatible, andmay also be biodegradable; preferably the formulation provides arelatively constant level of modulating agent release. The amount ofmodulating agent contained within a sustained release formulationdepends upon the site of implantation, the rate and expected duration ofrelease and the nature of the condition to be treated or prevented.

Selectin modulators are generally present within a pharmaceuticalcomposition in a therapeutically effective amount. A therapeuticallyeffective amount is an amount that results in a discernible patientbenefit, such as increased healing of a condition associated with excessselectin-mediated function (e.g., intercellular adhesion), as describedbelow.

In general, the modulating agents and compositions described herein maybe used for enhancing or inhibiting a selectin-mediated function. Suchenhancement or inhibition may be achieved in vitro and/or in vivo in awarm-blooded animal, preferably in a mammal such as a human, providedthat a selectin-expressing cell is ultimately contacted with amodulator, in an amount and for a time sufficient to enhance or inhibitselectin-mediated function.

Within certain aspects, the present invention provides methods forinhibiting the development of a condition associated with aselectin-mediated function, such as intercellular adhesion. In general,such methods may be used to prevent, delay or treat such a condition. Inother words, therapeutic methods provided herein may be used to treat adisease, or may be used to prevent or delay the onset of such a diseasein a patient who is free of disease or who is afflicted with a diseasethat is not associated with a selectin-mediated function. For example,the therapeutic methods have uses that may include the arrest of cellgrowth, the killing of cells, the prevention of cells or cell growth,the delay of the onset of cells or cell growth, or the prolongation ofsurvival of an organism.

A variety of conditions are associated with a selectin-mediatedfunction. Such conditions include, for example, tissue transplantrejection, platelet-mediated diseases (e.g., atherosclerosis andclotting), hyperactive coronary circulation, acute leukocyte-mediatedlung injury (e.g., adult respiratory distress syndrome (ARDS)), Crohn'sdisease, inflammatory diseases (e.g., inflammatory bowel disease),autoimmune diseases (MS, myasthenia gravis), infection, cancer (andmetastasis), thrombosis, wounds (and wound-associated sepsis), burns,spinal cord damage, digestive tract mucous membrane disorders(gastritis, ulcers), osteoporosis, rheumatoid arthritis, osteoarthritis,asthma, allergy, psoriasis, septic shock, traumatic shock, stroke,nephritis, atopic dermatitis, frostbite injury, adult dyspnoea syndrome,ulcerative colitis, systemic lupus erythematosus, diabetes andreperfusion injury following ischaemic episodes. Selectin modulators mayalso be administered to a patient prior to heart surgery to enhancerecovery. Other uses include pain management, prevention of restinosisassociated with vascular stenting, and for undesirable angiogenesis,e.g., associated with cancer.

Selectin modulators of the present invention may be administered in amanner appropriate to the disease to be treated (or prevented).Appropriate dosages and a suitable duration and frequency ofadministration may be determined by such factors as the condition of thepatient, the type and severity of the patient's disease and the methodof administration. In general, an appropriate dosage and treatmentregimen provides the modulating agent(s) in an amount sufficient toprovide therapeutic and/or prophylactic benefit. Within particularlypreferred embodiments of the invention, a selectin modulator may beadministered at a dosage ranging from 0.001 to 1000 mg/kg body weight(more typically 0.01 to 1000 mg/kg), on a regimen of single or multipledaily doses. Appropriate dosages may generally be determined usingexperimental models and/or clinical trials. In general, the use of theminimum dosage that is sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using assays suitable for the condition being treated orprevented, which will be familiar to those of ordinary skill in the art.

Selectin modulators may also be used to target substances to cells thatexpress a selectin. Such substances include therapeutic agents anddiagnostic agents. Therapeutic agents may be a molecule, virus, viralcomponent, cell, cell component or any other substance that can bedemonstrated to modify the properties of a target cell so as to providea benefit for treating or preventing a disorder or regulating thephysiology of a patient. A therapeutic agent may also be a prodrug thatgenerates an agent having a biological activity in vivo. Molecules thatmay be therapeutic agents may be, for example, polypeptides, aminoacids, nucleic acids, polynucleotides, steroids, polysaccharides orinorganic compounds. Such molecules may function in any of a variety ofways, including as enzymes, enzyme inhibitors, hormones, receptors,antisense oligonucleotides, catalytic polynucleotides, anti-viralagents, anti-tumor agents, anti-bacterial agents, immunomodulatingagents and cytotoxic agents (e.g., radionuclides such as iodine,bromine, lead, palladium or copper). Diagnostic agents include imagingagents such as metals and radioactive agents (e.g., gallium, technetium,indium, strontium, iodine, barium, bromine and phosphorus-containingcompounds), contrast agents, dyes (e.g., fluorescent dyes andchromophores) and enzymes that catalyze a colorimetric or fluorometricreaction. In general, therapeutic and diagnostic agents may be attachedto a selectin modulator using a variety of techniques such as thosedescribed above. For targeting purposes, a selectin modulator may beadministered to a patient as described herein. Since selectins areexpressed on endothelial cells involved in the formation of newcapillaries during angiogenesis, a selectin modulator may be used totarget a therapeutic agent for killing a tumor's vasculature. A selectinmodulator may also be used for gene targeting.

Selectin modulators may also be used in vitro, e.g., within a variety ofwell known cell culture and cell separation methods. For example,modulators may be linked to the interior surface of a tissue cultureplate or other cell culture support, for use in immobilizingselectin-expressing cells for screens, assays and growth in culture.Such linkage may be performed by any suitable technique, such as themethods described above, as well as other standard techniques.Modulators may also be used, for example, to facilitate cellidentification and sorting in vitro, permitting the selection of cellsexpressing a selectin (or different selectin levels). Preferably, themodulator(s) for use in such methods are linked to a detectable marker.Suitable markers are well known in the art and include radionuclides,luminescent groups, fluorescent groups, enzymes, dyes, constantimmunoglobulin domains and biotin. Within one preferred embodiment, amodulator linked to a fluorescent marker, such as fluorescein, iscontacted with the cells, which are then analyzed by fluorescenceactivated cell sorting (FACS).

Modulating agents as described above are capable, for example, ofinhibiting selectin-mediated cell adhesion. This ability may generallybe evaluated using any of a variety of in vitro assays designed tomeasure the effect on adhesion between selectin-expressing cells (e.g.,adhesion between leukocytes or tumor cells and platelets or endothelialcells). For example, such cells may be plated under standard conditionsthat, in the absence of modulator, permit cell adhesion. In general, amodulator is an inhibitor of selectin-mediated cell adhesion if contactof the test cells with the modulator results in a discernible inhibitionof cell adhesion. For example, in the presence of modulators (e.g.,micromolar levels), disruption of adhesion between leukocytes or tumorcells and platelets or endothelial cells may be determined visuallywithin approximately several minutes, by observing the reduction ofcells interacting with one another.

All compounds of the present invention or useful thereto, includephysiologically acceptable salts thereof.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Synthesis of BASA (FIG. 1A)

Synthesis of compound 4: Nitration of commercially available 2 (1 g) isaccording to the procedure described (for literature conditions see U.S.Pat. No. 4,534,905; Allison, F. et al. Helv. Chim. Acta 4:2139 (1952)).

The crude product 3 is dissolved in water (40 mL) and 10% Pd/C (0.3 g)added. The mixture is hydrogenated (˜45 psi) at room temperature for 48h. The catalyst is filtered through Celite and the filter bed is washedwith water. The filtrate is concentrated under vacuum to afford a pinksolid. After removal of the catalyst, the filtrate is concentrated to 15mL and an equal volume of ethanol is added. The precipitate is collectedby filtration to give compound 4 with very little impurity.

Synthesis of compound 7a: A solution of 5 (5 g) and 8 (4.45 g, 24.7mmol), and K₂CO₃ (2 M in H₂O, 24.7 mL, 49.4 mmol) in 10:1toluene/ethanol (70 mL) is treated with Pd(PPh₃)₄ (1.43 g, 1.24 mmol)and the mixture is refluxed for 20 h. After work up, recrystallizationof the crude product in EtOH and chromatographic purification of therecrystallization filtrate affords compound 9 (2.9 g, 46%, >90% HPLC)and 2.2 g of recovered 5. The product is characterized by ¹H NMR.

A mixture of 9 (2.9 g, 11.3 mmol) and LiOH.H₂O (1.43 g, 34.1 mmol) in1:1 THF/H₂O (250 mL) is stirred at RT for 21 h. The reaction affords 7(2.58 g, 94%, >90% HPLC) after work up. The product is characterized by¹H NMR.

DMF (20 μl) is added to a suspension of 7 (500 mg, 1.94 mmol), SOCl₂(0.23 mL, 3.10 mmol) and toluene (3 mL) and then heated to 80° C. After20 h the reaction is worked up to afford the acid chloride (640 mg). Theproduct 7a is characterized by IR and ¹H NMR.

Synthesis of compound 10: To a solution of amine 4 (268 mg, 0.641 mmol)in H₂O (2 mL) and dioxane (18 mL) is added a solution of 7a (273 mg,0.99 mmol) in dioxane (16 mL) dropwise over 30 min. The pH of thereaction mixture is adjusted to 8.5 with 0.25 M NaOH as the additionprogresses. The reaction is stirred at room temperature for 2.5 h afterthe addition. Purification by column chromatography (methanol/toluene1:1) followed by prep. TLC (methanol/toluene 1:1) affords 50 mg ofcompound 10, which is characterized by ¹H NMR and MS.

Hydrogenation of compound 10: A suspension of 10 (30 mg, 0.049 mmol) and10% Pd on carbon (50 mg) in H₂O (20 mL) is hydrogenated (55 psi) at roomtemperature for 4 h to yield the BASA of FIG. 1A.

Example 2 Synthesis of BASA (FIG. 1B)

Synthesis of compound 4: 3-nitro-benzyl iodide (1) (48.3 g) is added toan aqueous solution (pH 11) of commercially available,8-aminonaphthalene-1,3,5-trisulfonic acid (2) (29.5 g) with stirring atroom temperature. pH of the solution is adjusted to 1 and afterevaporation of the solvent, the product 3 (6.4 g) is precipitated outfrom ethanol.

Platinum catalyzed hydrogenation of compound 3 affords compound 4 (theBASA of FIG. 1B) in 96% yield.

Example 3 Synthesis of BACA (FIG. 2)

A suspension of 1 (8.9 g), paraformaldehyde (8.9 g), and H₂SO₄ (125 mL)is heated to 90° C. for 14 h and affords crude 2 (7.8 g) after work up.The crude product is 77% pure by HPLC and characterized by ¹H NMR.

To a solution of 2 (1.0 g) in acetone (30 mL) is added K₂CO₃ (3.1 g) anddimethylsulfate (1.4 mL) and the reaction is heated to reflux for 24 h.The reaction is combined with the next batch for work up andpurification.

To a solution of 2 (7.5 g) in acetone (225 mL) is added K₂CO₃ (23.2 g)and dimethylsulfate (10.8 mL) and the reaction is heated to reflux for16 h. The reaction, combined with the previous batch, affords 3 (7.3 g,74%) after work up and column chromatographic purification (ethylacetate/heptane 1:9). The product is 80% pure by HPLC and characterizedby ¹H NMR.

Chromic anhydride (6.94 g) is added to a suspension of 3 (7.16 g) inacetic anhydride (175 mL) at 3° C. and then stirred at room temperaturefor 15 h. The reaction affords 4 (5.89 g) after work up and columnpurification (100% dichloromethane). The product is 90% pure by HPLC andcharacterized by ¹H NMR.

To a suspension of 4 (5.89 g) in THF/H₂O (300 mL, 1:1) is added LiOH H₂O(1.74 g) at room temperature and the resulting mixture is stirred for 14h. After an acid/base work up, the product is obtained as a white solid.The product is dried under high vacuum and characterized by nmr and massspectroscopy.

Example 4 Synthesis of Glycomimetic (FIG. 3)

Synthesis of intermediate II: (−)-Shikimic acid (20 g) in MeOH (200 ml)and sulfuric acid (2 ml, 98%) are stirred at rt for 50 h. The reactionmixture is neutralized with 2N aqueous NaOH in the cold. Afterevaporation to dryness, the residue is purified by silica gelchromatography to afford II (19.2 g).

Synthesis of intermediate (III): Methyl shikimate (II, 10 g), 2,2dimethoxypropane (10 ml) and p-TsOH (0.8 g) are dissolved inacetonitrile (125 ml) and stirred at rt for 1 h. The reaction mixture isthen neutralized with triethylamine (2 ml) and evaporated to dryness.The residue is chromatographed on silica gel to yield III (11 g).

Synthesis of intermediate IV: The shikimic acid derivative III (10 g)and PtO₂/C (10%, 250 mg) in MeOH (40 ml) are hydrogenated at rt undervigorous stirring. After 16 h the reaction mixture is filtered overcelite and evaporated to dryness. The residue is chromatographed onsilica gel to yield IV.

Synthesis of intermediate V: To a solution of IV (8 g) in DCM (100 ml)at 0° C. are added pyridine (12 ml), acetic anhydride (7 ml) and a DMAP(25 mg). The reaction mixture is stirred at rt for 1 h, and diluted withEtOAc (250 ml). After washing with 0.5 M aqueous HCl (3×50 ml),saturated solution of KHCO₃ (3×50 ml) and brine (3×50 ml), the combinedorganic layers are dried (Na₂SO₄) and evaporated to dryness. The residueis purified by chromatography on silica gel to yield V (6.8 g).

Synthesis of intermediate VI: A solution of V (6.0 g) in acetic acid (30ml, 80%) is stirred at 80° C. for 1 h. Solvent is evaporated off and theresidue is purified by chromatography on silica gel (DCM/MeOH 14:1) toyield VI (3.6 g).

Synthesis of intermediate (VII): A solution of VI (3 g) and p-TsCl (3.5g) in pyridine (30 ml) is stirred at rt for 6 h. MeOH (5 ml) is addedand the solvent is evaporated at reduced pressure, the residue dissolvedin EtOAc (3×150 ml) and the organic layers are washed with 0.5 M aqueousHCl (0° C.), water (cold) and brine (cold). The combined organic layersare dried (Na₂SO₄), filtered on Celite and evaporated to dryness. Theresidue is purified by chromatography on silica gel (toluene/EtOAc 4:1)to yield VII (3.7 g).

Synthesis of compound VIII: A solution of VII (3 g) and NaN₃ (2.5 g) inDMF (20 ml) is stirred at 80° C. The reaction mixture is cooled to rtand diluted with EtOAc (200 ml) and water (50 ml). The organic layer isadditionally washed twice with water (2×50 ml) and once with brine (50ml). All aqueous layers are extracted twice with EtOAc (2×50 ml). Thecombined organic layers are dried with Na₂SO₄, filtered and the solventis evaporated off. The residue is purified by chromatography on silicagel (petroleum ether/EtOAc 5:2) to give VIII (2.2 g).

Synthesis of compound X: To a solution of ethyl2,3,4-tri-O-benzyl-α-L-fucothiopyanoside IX (1.5 g) in DCM (3 ml),bromine (150 μl) is added at 0° C. under argon. After 5 min the coolingbath is removed and the reaction mixture is stirred for additional 25min at rt. Cyclohexene (200 μl) is added and the reaction mixture isadded to a solution of VIII (400 mg), (Et)₄NBr (750 mg) and powdered 4 Åmolecular sieves in DCM (10 ml) and DMF (5 ml). After 16 h,triethylamine (1.5 ml) is added and stirred for an additional for 10min, diluted with EtOAc (50 ml) and washed with sat. aqueous NaHCO₃,water and brine. The aqueous layers are extracted twice with EtOAc (2×50ml). The combined organic layers are dried (Na₂SO₄), filtered andevaporated to dryness. The residue is purified by chromatography onsilica gel (toluene/EtOAc 9:1) to yield X (700 mg).

Synthesis of compound XI: To a solution of X (1.5 g) in MeOH (20 ml) isadded freshly prepared NaOMe (80 mg) and the reaction mixture is stirredin a pressure tube at 80° C. for 20 h. The reaction mixture is cooled tort and neutralized with acetic acid. Solvent is evaporated to drynessand the residue is dissolved in ether. Freshly prepared diazomethane isadded and the excess diazomethane is neutralized with acetic acid.Solvent is evaporated off to give XI (1.25 g).

Synthesis of building block XV: This synthesis is done exactly in sameway as described previously (Helvetica Chemica Acta 83:2893-2907(2000)).

Synthesis of compound XVI: A mixture of XI (1.6 g), XV (3 g) andactivated powdered molecular sieves 4 Å (1 g) in DCM (17 ml) is stirredat rt under argon for 2 h. Then DMTST (2 g) is added in 4 equal portionsover a period of 1.5 h. After 24 h the reaction mixture is filtered overCelite and the filtrate is diluted with DCM (100 ml). The organic layeris washed with sat. aqueous NaHCO₃ and brine and the aqueous layers areextracted twice with DCM. The combined organic layers are dried(Na₂SO₄), filtered and evaporated to dryness. The residue is purified bychromatography on silica gel (toluene/EtOAc 8:1) to yield XVI (1.5 g).

Synthesis of compound XVII: To a solution of XVI (500 mg) and oroticacid chloride (500 mg) in dichloromethane (10 ml) is added a solution oftriphenylphosphine (500 mg in 5 ml dichloromethane) dropwise during 10min. The reaction mixture is stirred at rt for 25 h and the solvent isevaporated off. The residue is purified (chromatography on silica gelDCM/MeOH 19:1) to give XVII (250 mg).

Synthesis of compound XVIII: To a solution of XVII (200 mg) indioxane-water (5:1, 12 ml) is added 10% Pd—C (100 mg) and the reactionmixture is stirred vigorously under hydrogen (55 psi) for 24 h. Catalystis filtered through a bed of celite and the solvent is evaporated off.Residue is purified by silica gel chromatography to give compound XVIII(150 mg).

Synthesis of XIX: To a solution of compound XVIII (145 mg) in MeOH (5ml) is added a solution of NaOMe in MeOH (25%, 0.025 ml) and thereaction mixture is stirred at rt for 4 h, neutralized with acetic acidand the solvent is evaporated off. Residue is dissolved in water andpassed through a bed of Dowex 50wX-8 (Na-form) resin. Water wash isevaporated off to afford compound XIX (100 mg).

Synthesis of EDA-XIX: XIX (80 mg) is heated at 70° C. withethylenediamine (EDA) (1 ml) with stirring for 5 h. Solvent isevaporated off and the purified by sephadex G-25 column to give EDA-XIX(82 mg).

Example 5 Synthesis of Glycomimetic (FIG. 4)

Synthesis of compound XXI: To a solution of compound XX (1.5 g,synthesized according to previously published procedure CarbohydrateChemistry and Biochemistry, 2000, vol. 1, page 345-365) in pyridine (60ml) is added benzoic anhydride (0.73 g) and dimethyl amino pyridine(0.02 g). The reaction mixture is stirred at room temperature for 20 h.Solvent is evaporated off and the residue is dissolved indichloromethane. The solution is washed successively with cold 1N HCland water. The solution is dried (sodium sulfate) and concentrated todryness. Residue is purified by column chromatography (silica gel) togive compound XXI (1 g).

Synthesis of compound XXII: To a solution of compound XXI indichloromethane (20 ml) is added trifluoroacetic acid (20 ml) at 0° C.and the reaction mixture is stirred at the same temperature for 1 h.Solvent is evaporated off and the residue is purified by columnchromatography (silica gel) to give compound XII (0.6 g).

Synthesis of compound XXIII: To a solution of compound XXII (1 g) indichloromethane (40 ml) is added DBU (0.05 ml) and tricholroacetonitrile(0.4 g) at 0° C. The solution is stirred at the same temperature for 1.5h. Solvent is evaporated off and purified by column chromatography(silica gel) to give compound XXIII (0.6 g).

Synthesis of compound XXIV: To a mixture of compound XI (0.7 g),compound XXIII (0.5 g) in dichloromethane (40 ml) and molecular sieves(4 Å, 5 g) is added a solution of TMSOTf (0.15 ml in dichloromethane (5ml)) dropwise at 0° C. with stirring. Stirring is continued at the sametemperature for 2 h. Triethylamine (0.2 ml) is added and the reactionmixture is filtered through a bed of celite. Reaction mixture is washedwith cold saturated solution of sodium bicarbonate and water. Dried(sodium sulfate) and concentrated to dryness. The residue is purified bycolumn chromatography (silica gel) to give compound XXIV (0.7 g).

Synthesis of compound XXV: To a solution of compound XXIV (0.6 g) in DMF(30 ml) is added DBU (30 drops) and dl-dithio-threitol (DTT, 0.28 g).The reaction mixture is stirred at room temperature for 1 h. Solvent isevaporated off, residue is dissolved in dichloromethane and washed withwater. Organic layer is dried (sodium sulfate) and concentrated todryness. The residue is purified by column chromatography (silica gel)to give compound XXV (0.45 g).

Synthesis of compound XXVI: To a solution of compound XXV (0.4 g) in DMF(10 ml) is added orotic acid (0.14 g), EEDQ (0.19 g),4-methyl-morpholine (0.09 g) and the reaction mixture is stirred at 70°C. for 20 h. Solvent is evaporated off and residue is dissolved indichloromethane. The solution is washed with cold saturated sodiumbicarbonate solution and water. Organic layer is dried (sodium sulfate)and concentrated to dryness. The residue is purified by columnchromatography (silica gel) to give compound XXVI (0.2 g).

Synthesis of compound XXVII: Compound XXVII (0.2 g) is hydrogenatedexactly in the same condition as described and the intermediate ispartially debenzoylated using NaOMe in MeOH as described to givecompound XXVII (0.050 g) after chromatographic purification.

Synthesis of compound XXVIII: Compound XXVII is treated withethylenediamine as described and purified by column chromatography(silica gel and gel filtration sephadex G-25) to give compound XXVIII(25 mg).

Example 6 Synthesis of PEGylated BASA (FIG. 7B)

To a solution of 3,6-dioxaoctanedioic acid (PEG, 200 mg, availablecommercially) in DMF (1 ml) is added Hunig base (0.4 ml), and then HATU(0.35 g) is added after 5 min. The solution is stirred at RT for 10 min.and then a solution of the BASA of Example 2 (50 mg) in DMF (0.1 ml) isadded. The reaction mixture is stirred for 4 h at rt and the solvent isevaporated off. The residue is purified by hplc (reverse-phase C18column) to give XXXIII (40 mg).

Example 7 Synthesis of PEGylated BASA (FIG. 7A)

This synthesis is performed in the same way as described in Example 6,except using the BASA of Example 1 to give XXXII (50 mg).

Example 8 Synthesis of PEGylated BACA (FIG. 7C)

Synthesis of intermediate XXXIV (Method 1): The BACA of Example 3 (0.5g) is suspended in methanol-water (1 ml, 9:1) and the pH is adjusted to8.2 by the addition of an aqueous solution of Cs₂CO₃. The solvent isremoved and then coevaporated with toluene. The residue is dissolved inDMF (1 ml). Benzylbromide (0.5 ml) is added and stirred for 20 h at roomtemperature. Dichloromethane (15 ml) is added washed with cold water.Organic layer is dried (anhydrous sodium sulfate) and solvent isevaporated off. The residue is purified by column chromatography(silica) to give XXXIV (0.48 g).

Synthesis of intermediate XXXIV (Method 2): To a solution of the BACA ofExample 3 (1 g) in DMF is added N,N-diisopropyl ethylamine (1.5 g) andBenzylbromide (1.5 g). The reaction mixture is stirred at 50° C. for 20h. Solvent is evaporated off and the residue is purified by columnchromatography (silica) to give XXXIV.

Synthesis of intermediate of XXXV: To a solution of XXXIV (0.2 g) inMeOH (10 ml) is added sodiumborohydride (0.070 g) at 0° C. and thereaction mixture is stirred at 0° C. for 1 h. The reaction is quenchedby addition of acetic acid and concentrated to dryness. Residue ispurified column chromatography (silica) to afford XXXV (0.16 g).

Synthesis of intermediate of XXXVb: To a solution of XXXV (0.36 g) indichloromethane is added triethylamine (0.6 ml) and MeSO₂Cl (0.29 ml).The reaction mixture is stirred at RT for 21 h. Reaction mixture isdiluted with dichloromethane, washed with water, 1 M HCl, and brine.Organic layer is dried (Na₂SO₄) and concentrated to dryness to givecrude XXXVa. To a solution of crude XXXVa in DMF (5 ml) is added NaN₃(0.18 g). The reaction mixture is stirred at 100° C. for 4 h and thesolvent is evaporated off. The residue is dissolved in CH₂Cl₂ and washedwith cold brine, cold 1 M HCl, cold NaHCO₃ solution, and cold water.Organic layer is dried (Na₂SO₄) and concentrated to dryness. The residueis purified by column chromatography (silica) to give XXXVb (0.14 g).

Synthesis of intermediate XXXVc: To a solution of XXXVc (0.08 g) in DMF(3 ml) is added DTT (0.04 g) and DBU (0.02 ml) and stirred at RT for 1h. Solvent is evaporated off and the residue is dissolved in EtOAc,washed with H₂O and the organic layer is concentrated to dryness. Theresidue is purified by column chromatography (silica) to affordintermediate XXXVc (0.061 g).

Synthesis of intermediate XXXVd: To a solution of monoprotectedPEG-dicarboxylic acid (0.6 g) in DMF (3 ml) is addeddiisopropylethylamine (0.24 ml) and HATU (0.513 g) with stirring. Asolution of intermediate XXXVc (0.185 g) in DMF (3 ml) is added into theabove solution. The reaction mixture is stirred at RT for 1 h. Solventis evaporated off and the residue is dissolved in EtOAc. EtOAc layer iswashed with water and purified by column chromatography to giveintermediate XXXVd.

Synthesis of XXXVI: To a solution of XXXVd (0.185 g) in glacial AcOH (3ml) is added Zn dust (0.1 g) and the reaction is stirred at 40° C. for30 min. Reaction mixture is filtered through a celite bed and Zn cake iswashed with MeOH. The filtrate is concentrated to dryness and purifiedby sep-pak C18 column to afford XXXVI (0.050 g).

Synthesis of intermediate XXXIVa: A suspension of TiCl₄-Tetrahydrofurancomplex (0.705 g) and Zn-dust (0.28 g) in THF (25 ml) is refluxed for 2h at 75° C. for 2 h with stirring under inert atmosphere. To thismixture is added a solution of the BACA of Example 3 (0.3 g) andN-fluorenylmethoxycarbonyl-3-aminopropanol (0.312 g, prepared asdescribed in the literature Casimiro-Garcia et al, Bioorg. Med. Chem.,1979 (2001) 2827). The reaction mixture is stirred at 75° C. for 2.5 h(McMurray coupling) under inert atmosphere. The reaction is cooled downto RT and H₂O (30 ml) is added, filtered through a celite bed and thefiltrate is washed three times with EtOAc (30 ml each). Organic layer iscollected together and dried (Na₂SO₄), filtered and concentrated todryness. Residue is purified by column chromatography to yield XXXIVa.

Synthesis of intermediate XXXIVb: To a solution of XXXIVa (0.444 g) inanhydrous THF (21 ml) is added piperidine (6.25 ml) and the reactionmixture is stirred at RT for 3 h. The solvent is evaporated off and theresidue is purified by column chromatography (silica) to giveintermediate XXXIVb (0.26 g).

Solid phase synthesis of intermediate XXXIVc: A mixture ofPS-Carbodiimide resin (0.200 g), HOBt (0.030 g), and mono protectedPEG-COOH (0.080 g) in CH₂Cl₂ (3 ml) is stirred for 5 min at RT in asyringe reactor. To the above mixture is added a solution ofintermediate XXXIVb (0.080 g) in CH₂Cl₂ (3 ml) and the reaction mixtureis stirred at RT for 3 h. MP-carbonate resin (0.216 g) is added andstirred for 2 h at RT. Resin is filtered off and then the resin iswashed 5 times with CH₂Cl₂. Filtrate is combined and concentrated todryness to afford intermediate XXXIVc.

Synthesis of intermediate XXXVIa: XXXIVc (0.12 g) is treated withZn/AcOH exactly in the same as described for the synthesis ofintermediate XXXVI to yield intermediate XXXVIa (0.104 g).

Example 9 Synthesis of PEGylated BACA (FIG. 7C)

Synthesis of intermediate XXXIVa: A suspension of TiCl₄-Tetrahydrofurancomplex (0.705 g) and Zn-dust (0.28 g) in THF (25 ml) is refluxed for 2h at 75° C. for 2 h with stirring under inert atmosphere. To thismixture is added a solution of the BACA of Example 3 (0.3 g) andN-fluorenylmethoxycarbonyl-3-aminopropanol (0.312 g, prepared asdescribed in the literature Casimiro-Garcia et al, Bioorg. Med. Chem.,1979 (2001) 2827). The reaction mixture is stirred at 75° C. for 2.5 h(McMurray coupling) under inert atmosphere. The reaction is cooled downto RT and H₂O (30 ml) is added, filtered through a celite bed and thefiltrate is washed three times with EtOAc (30 ml each). Organic layer iscollected together and dried (Na₂SO₄), filtered and concentrated todryness. Residue is purified by column chromatography to yield XXXIVa.

Synthesis of intermediate XXXIVb: To a solution of XXXIVa (0.444 g) inanhydrous THF (21 ml) is added piperidine (6.25 ml) and the reactionmixture is stirred at RT for 3 h. The solvent is evaporated off and theresidue is purified by column chromatography (silica) to giveintermediate XXXIVb (0.26 g).

Solid phase synthesis of intermediate XXXIVc: A mixture ofPS-Carbodiimide resin (0.200 g), HOBt (0.030 g), and mono protectedPEG-COOH (0.080 g) in CH₂Cl₂ (3 ml) is stirred for 5 min at RT in asyringe reactor. To the above mixture is added a solution ofintermediate XXXIVb (0.080 g) in CH₂Cl₂ (3 ml) and the reaction mixtureis stirred at RT for 3 h. MP-carbonate resin (0.216 g) is added andstirred for 2 h at RT. Resin is filtered off and then the resin iswashed 5 times with CH₂Cl₂. Filtrate is combined and concentrated todryness to afford intermediate XXXIVc.

Synthesis of XXXVIa: XXXIVc (0.12 g) is treated with Zn/AcOH exactly inthe same as described for the synthesis of intermediate XXXVI to yieldintermediate XXXVIa (0.104 g).

Example 10 Synthesis of Glycomimetic-BASA (FIG. 8A)

To a solution of XXXII from Example 7 (0.015 g) in DMF (0.1 ml) is addedHunig base (0.015 ml) and then HATU (0.007 g). The reaction mixture isstirred for 10 min at RT. A solution of EDA-XIX from Example 4 (0.010 gin DMF ml) is added and the reaction mixture is stirred at RT for 8 h.Solvent is evaporated off and the residue is purified by sephadex G-25chromatography to give Glycomimetic-BASA of FIG. 8A (0.008 g).

Example 11 Synthesis of Glycomimetic-BACA (FIG. 8B)

Coupling between EDA-XIX and XXXVI: To a solution of XXXVI from Example8 in DMF is added diisopropylethylamine and then HATU. The solution isstirred for 3 min at RT. The above solution is then added to EDA-XIXwith stirring in a conical vial. Reaction mixture is stirred for 2 h atRT. Solvent is evaporated off to give crude intermediate XXXVIb and isused for the next step without further purification.

The crude XXXVIb is treated with NaOMe-MeOH—H₂O for 2 h and thenpurified by gel filtration to give a Glycomimetic-BACA.

Coupling between EDA-XIX and XXXVIa from Example 9: This couplingreaction is performed exactly in the same way as described for thesynthesis of XXXVIb to give crude product XXXVIc.

XXXVIc is treated with NaOMe-MeOH—H₂O exactly in the same way asdescribed to afford a Glycomimetic-BACA.

Example 12 Synthesis of Glycomimetic-BASA (FIG. 8C)

This synthesis is performed in the same way as described in Example 10using XXXIII from Example 6 and EDA-XIX from Example 4 to giveGlycomimetic-BASA of FIG. 8C. Alternatively, XXXIII can be replaced withXLV from Example 18 for reaction with EDA-XIX.

Example 13 Synthesis of Glycomimetic-BASA (FIG. 9A)

This synthesis is performed in the same way as described in Example 10using XXXII from Example 7 and EDA-XXVIII from Example 5 to giveGlycomimetic-BASA of FIG. 9A.

Example 14 Synthesis of Glycomimetic-BACA (FIG. 9B)

Coupling between EDA-XXVIII and XXXVI: This coupling reaction isperformed exactly in the same way as described for the synthesis ofXXXVIb to give crude product XXXVId.

XXXVId is treated with NaOMe-MeOH—H₂O exactly in the same way asdescribed to afford a Glycomimetic-BACA.

Coupling between EDA-XXVIII and XXXVIa: This coupling reaction isperformed exactly in the same way as described for the synthesis ofXXXVIb to give crude product XXXVIe.

XXXVIe is treated with NaOMe-MeOH—H₂O exactly in the same way asdescribed to afford a Glycomimetic-BACA.

Example 15 Synthesis of Glycomimetic-BASA (FIG. 9C)

This synthesis is performed in the same way as described in Example 10using XXXIII from Example 6 and EDA-XXVIII from Example 5 to giveGlycomimetic-BASA of FIG. 9C. Alternatively, XXXIII can be replaced withXLV from Example 18 for reaction with EDA-XXVIII.

Example 16 Synthesis of Glycomimetic (FIG. 12)

Synthesis of intermediate XXXVII: A solution of intermediate XXIV (0.1g) from Example 5 in pyridine is treated with nicotinyl chloride (0.08ml) in pyridine and dimethylaminopyridine (0.04 g) in the same way asdescribed in Example 5 for the synthesis of intermediate XXV to giveintermediate XXXVII (0.075 g).

Synthesis of intermediate XXXVIII: Intermediate XXXVII (0.07 g) istreated with triphenylphosphine and orotic acid chloride in the same wayas described in Example 4 for the synthesis of compound XVII to giveXXXVIII (0.048 g).

Synthesis of intermediate XXXIX: Hydrogenation of intermediate XXXVIII(0.04 g) with Pd/C as described gives compound XXXIX (0.02 g).

Synthesis of Compound XL: Intermediate XXXIX (0.015 g) is treated with0.5N NaOH for 10 min at 60° C. to afford compound XL (0.010 g) afterpurification by sephadex G-25 gel filtration.

Example 17 Synthesis of Glycomimetic (FIG. 13)

Synthesis of XLI: To a suspension of compound XVI (0.1 g) int-BuOH-water (4 ml, 1:1) is added 1-ethynyl-3-fluorobenzene (0.9 g), 1%CuSO4 (0.1 ml), and Na—I-ascorbate (4 mg). The mixture is heated (70°C.) with stirring for 20 h. Solvent is evaporated off and the residue isdissolved in dichloromethane. Organic layer is washed with water dried(anhydrous sodium sulfate) and concentrated to dryness. The residue ispurified by column chromatography (silica gel) to give compound XLI(0.08 g).

Synthesis of XLII: Compound XLI (0.25 g) is dissolved in dioxane-water(4:1, 7.5 ml). 10% Pd—C (0.25 g) is added, followed by AcOH (7 drops).The mixture is hydrogenated for 15 h at 40 psi. The reaction mixture isfiltered through a celite bed and concentrated to dryness to givecompound XLII (0.2 g).

Synthesis of XLIII: To a solution of compound XLII (0.2 g) in MeOH (5ml) is added a solution of NaOMe in MeOH (0.05 ml) and the reactionmixture is stirred at room temperature for 2 h. The reaction mixture isneutralized with few drops of acetic acid and concentrated to dryness.Residue is purified by column chromatography (silica gel) to givecompound XLIII (0.15 g).

Synthesis of XLIV: Compound XLIII (0.15 g) is dissolved inethylenediamine (7 ml) and the reaction mixture is stirred at 70° C. for9 h. Solvent is evaporated off and the residue is first purified bycolumn chromatography (silica gel) and then by reverse phase C18 to givecompound XLIV (0.11 g).

Synthesis of Glycomimetic-BASA (compound XLV): This synthesis isperformed in the same way as described in example 10 using XXXIII fromexample 6 and XLIV to give compound XLV.

Example 18 Synthesis of Glycomimetic-BASA (FIG. 14)

Synthesis of compound XLV: To a solution of 3,6-dioxaoctanedioic acid(PEG, 200 mg, available commercially) in DMF (1 ml) is added Hunig base(0.4 ml) and then HATU (0.35 g) is added after 5 min. The solution isstirred at RT for 10 min and then solution of8-aminonaphthalene-1,3,6-trisulfonic acid (50 mg, availablecommercially) in DMF is added. The reaction mixture is stirred for 4 hat RT and the solvent is evaporated off. The residue is purified by hplc(reverse-phase C18 column) to give XLV (25 mg).

Synthesis of compound XLVI: This synthesis is performed in the same wayas described in example 10 using XLV and EDA-XIX from example 4 to givecompound XLVI (4 mg).

Example 19 Synthesis of Glycomimetic-BASA (FIG. 15)

Synthesis of XLVII: Starting with compound XXIV, this synthesis isperformed in the same way as described for the synthesis of XLI to givecompound XLVI.

Synthesis of XLVIII: Starting with compound XLVII, this synthesis isperformed in the same way as described for compound XLIII (from XLI) togive compound XLVIII.

Synthesis of XLIX: Starting with compound XLVIII, this synthesis isperformed in the same way as described for the synthesis of XLIV to givecompound XLIX.

Synthesis of Glycomimetic-BASA (compound L): This synthesis is performedin the same way as described in example 10 using XXXIII from example 6and XLIX to give compound L. Alternatively, XXXIII can be replaced withXLV from Example 18 for reaction with XLIX.

Example 20 Synthesis of Glycomimetics (FIG. 16)

Synthesis of compound I: as described in the literature [J. Org. Chem.54, 3738-3740 (1989); Liebigs Annalen der Chemie 575, 1 (1952)]

Synthesis of intermediate II: I (2.8 g, 15.04 mmol), pyridine (4.8 ml,60.15 mmol), benzoylchloride (3.5 ml, 30.07 mmol) and a catalyticalamount of dimethyl aminopyridine are stirred in dichloromethane (6 ml)at room temperature (“r.t.”). After 2 h, TLC control shows completion ofthe reaction. The reaction mixture then is diluted with ethyl acetate(200 ml) and washed with water, 1N aqueous HCl (ice-cooled), saturatedaqueous NaHCO₃ and brine (each 50 ml). The aqueous layers are washedtwice with ethyl acetate (2×150 ml), combined and dried with Na₂SO₄.After filtration and evaporation of the solvent the residue is purifiedby chromatography on silica gel (PE/EtOAc 4:1) to yield compound II(3.78 g, 86%).

Synthesis of intermediate III: II (3.78 g, 13.02 mmol) and NaBH₄ arestirred in methanol (35 ml) at 0° C. After 30 min. the reaction mixtureis quenched with water (15 ml) and neutralized with 1N aqueous AcOH.Again water (10 ml) is added and the mixture is extracted 3 times withdichloromethane (3×150 ml). The combined organic layers are dried withNa₂SO₄, filtered and evaporated. Chromatography of the residue on silicagel (PE/EtOAc 3.2) gives compound III (3.7 g, 97%). [α]_(D)+79.78°(c=0.940, CH₂Cl₂);

Synthesis of intermediate IV: To a solution of III (3.4 mg, 11.64 mmol)in CHCl₃ (50 ml, filtered on basic Alox) at r.t. under argon is added1-chloro-N,N,2-trimethylpropenylamine (4.94 ml, 34.93 mmol) by asyringe. The reaction mixture is stirred at reflux until TLC controlindicated completion of the reaction (30 min.) After cooling to r.t. thereaction mixture is quenched with triethylamine (6 ml) and evaporated(bath temperature 30° C.) to dryness. The residue is purified bychromatography on silica gel (PE/EtOAc 9:1) to yield compound IV (3.3 g,92%).

Synthesis of intermediate V: To a solution of IV (3.25 g, 10.47 mmol) indry toluene (40 ml) under argon are added freshly distilled Bu₃SnH(30.58 ml, 115.14 mmol) and AIBN (1.7 g, 10.47 mmol). The reaction isrefluxed. After 75 min., when TLC shows completion of the reaction, thereaction mixture is cooled to r.t. and then diluted with acetonitrile(50 ml). The solution is washed with hexane (50 ml) and the hexane layeris extracted again with acetonitrile (50 ml). The combined acetonitrilelayers are evaporated. Chromatography of the residue on silica gel(toluene/EtOAc 14:1) yields compound V (2.66 g, 92%). [α]_(D)−117.79°(c=1.810, CH₂Cl₂).

Synthesis of intermediate VI: A solution of V (2.63 g, 9.54 mmol) inaqueous AcOH 80% is stirred at 80° C. When TLC control indicatescompletion of the reaction (30 min.), reaction mixture is cooled to r.t.After neutralizing with aqueous NaOH, the mixture is extracted 3 timeswith dichloromethane (3×200 ml).

The combined organic layers are dried with Na₂SO₄, filtered andevaporated to dryness. The residue is purified by chromatography onsilica gel (PE/EtOAc 3:2) to yield compound VI (2.01 g, 89%).[α]_(D)−54.86° (c=1.420, CH₂Cl₂).

Synthesis of intermediate VII: A solution of VI (1.98 g, 8.39 mmol),freshly recrystallized toluene-4-sulfonylchloride (1.9 g, 10.07 mmol),Bu₂SnO (2.09 g, 8.39 mmol) and triethylamine (1.8 ml, 16.78 mmol) in drydichloromethane (40 ml) is stirred at r.t. under argon. After 20 h, TLCcontrol indicated completion of the reaction. The reaction mixture isthen quenched with methanol (15 ml) and then evaporated to dryness.Chromatography of the residue on silica gel (toluene/EtOAc 8:1) givescompound VII (2.65 g, 85%). [α]_(D)−68.08° (c=0.448, CH₂Cl₂).

Synthesis of intermediate VIII: A mixture of VII (640 mg, 1.71 mmol) andNaN₃ (555 mg, 8.55 mmol) in DMF (30 ml) is stirred under argon at 80° C.When TLC control shows completion of the reaction (after 1 h), thereaction mixture is cooled to r.t., diluted with dichloromethane (50 ml)and washed with water (50 ml). The aqueous layer is then extracted twicewith dichloromethane (2×50 ml) and the combined organic layers are driedwith Na₂SO₄, filtered and evaporated. The residue is purified bychromatography on silica gel (toluene/EtOAc 6:1) to yield compound VIII(391 mg, 87%). [α]_(D)−69.39° (c=2.330, CH₂Cl₂).

Synthesis of intermediate IX: To a stirred solution of ethyl 2,3,4-tri-O-benzyl-α-L-fucothiopyranoside (223 mg, 0.47 mmol) in drydichloromethane (1 ml) at 0° C. under Ar atmosphere, bromine (48μ, 0.54mmol) is added. After 5 min. the cooling bath is removed and thereaction mixture is stirred for additional 40 min. at r.t. To remove theexcess of bromine, cyclohexene (50 μl) is added, leading to adecolorization of the reaction mixture. Then the reaction mixture isadded to a pre-stirred solution (1 h, r.t.) of VIII (61 mg, 0.23 mmol),(Et)₄NBr (98 mg, 0.47 mmol) and powdered 4 Å molecular sieves (100 mg)in dichloromethane (1.6 ml) and DMF (1 ml). After 18 h, the reaction isquenched with pyridine (2 ml) and stirred for additional for 15 min.,before it is diluted with EtOAc (50 ml) and washed with sat. aqueousKHCO₃, water and brine (each 50 ml). The aqueous layers are extractedtwice with EtOAc (2×50 ml). The combined organic layers are dried withNa₂SO₄, filtered and evaporated to dryness. The residue is purified bychromatography on silica gel (toluene/EtOAc 9:1) to yield IX (116 mg,73%). [α]_(D)−95.96° (c=1.040, CH₂Cl₂).

Synthesis of intermediate X: IX (530 mg, 0.78 mmol) in MeOH (10 ml) anda catalytical amount of freshly prepared NaOMe are stirred at r.t. underAr for 48 h. The reaction mixture is neutralized with powderedAmberlyst-15 and filtered over Celite. The filtrate is evaporated todryness and purified by chromatography on silica gel (toluene/EtOAc 9:1)to give compound X (380 mg, 85%). [α]_(D)−76.425° (c=4.00, CH₂Cl₂).

Synthesis of intermediate XI: A mixture of X (184 mg, 0.32 mmol), thegalactose building block [375 mg, 0.4811 mmol, synthesis as describedpreviously, Helv. Chim. Acta 83: 2893-2907 (2000)] and activatedpowdered molecular sieves 4 Å (200 mg) in dichloromethane (3 ml) isstirred at r.t. under argon for 4 h. Then DMTST [B] (165 mg, 0.64 mmol)is added in 4 equal portions over a period of 1.5 h to a pre-stirredmixture (4 h, r.t.) of activated powered molecular sieves 4 Å (100 mg)in dichloromethane (3 ml). After 92 h, when TLC control shows completionof the reaction, the reaction mixture is filtered over Celite and thefiltrate is diluted with dichloromethane (50 ml). The organic layer iswashed with sat. aqueous NaHCO₃ and brine (each 20 ml) and the aqueouslayers are extracted twice with dichloromethane (2×50 ml). The combinedorganic layers are dried with Na₂SO₄, filtered and evaporated todryness. The residue is purified by chromatography on silica gel(toluene/EtOAc 9:1) to yield compound XI (310 mg, 75%). [α]_(D)−36.98°(c=2.32, CH₂Cl₂).

General Procedure A

Synthesis of intermediate XII: To a stirred solution of XI (100 mg,0.077 mol) in dry dichloromethane (3 ml), acetic acid chloride (27 μl,0.387 mmol) is added. After 5 min. PPh₃ (101 mg, 0.387 mmol) is addedand the solution is stirred at r.t. When TLC after 22 h shows completionof the reaction, the solvent is removed and the residue chromatographedon silica gel (toluene/EtOAc 2:1) yielding compound XII (43 mg, 43%).[α]_(D)−55.33° (c=2.185, CH₂Cl₂).

Synthesis of intermediate XIII: According to procedure A: XI (80 mg,0.062 mol) with orotic acid chloride (59 mg, 0.310 mmol) and PPh₃ (81mg, 0.310 mmol), 4 h, (dichloromethane/MeOH 25:1) gives compound XIII(35 mg, 40%). [α]_(D)−26.08° (c=1.48, CH₂Cl₂).

Synthesis of intermediate XIV: According to procedure A: XI (70 mg,0.054 mmol) with biphenyl-4-carboxylic acid chloride (58 mg, 0.271 mmol)and PPh₃ (71 mg, 0.271 mmol), 4 h, (toluene/EtOAc 6:1) gives XIV (22 mg,28%). [α]_(D)−32.11° (c=1.065, CH₂Cl₂).

Synthesis of intermediate XV: According to procedure A: XI (45 mg, 0.035mmol) with biphenyl-2-carboxylic acid chloride (125 mg. 0.577 mmol) andPPh₃ (151 mg, 0.577 mmol), 5 h, (toluene/EtOAc 6:1), gives compound XV(22 mg, 44%). [α]_(D)−19.54° (c=1.10, CH₂Cl₂).

General Procedure B

Synthesis of intermediate XVI: A mixture of XI (120 mg, 0.093 mmol) andPPh₃ (30 mg, 0.116 mmol) in CH₂Cl₂ (2 ml) and water (100 μl) is stirredfor 44 h at r.t. Then the solvents are removed and the residue dissolvedin CH₂Cl₂ (2 ml) and DIC (11.8 mg, 0.094 mmol) and the vanillic acid (24mg, 0.139 mmol) are added. After the mixture is stirred at r.t. foradditional 62 h, the solvents are removed and the crude product ispurified by chromatography on silica gel (toluene/EtOAc 2.5:1) to givecompound XVI (62 mg, 47%). [α]_(D)−29.58° (c=2.86, CH₂Cl₂).

General Procedure C

Synthesis of intermediate XVII: A mixture of XII (40 mg, 0.0306 mmol),Pd(OH)₂ (30 mg), dioxane (2 ml) and water (0.4 ml) is hydrogenated in aParr-shaker under 5 bar at r.t. After 20 h TLC control indicatedcompletion of the reaction. The reaction mixture is filtered over Celiteand evaporated to dryness. Purification of the crude product bychromatography (CH₂Cl₂/MeOH 9:1) yielded XVII (20 mg, 69%).[α]_(D)−43.50° (c=1.00, MeOH).

Synthesis of intermediate XVIII: According to procedure C: XIV (26 mg,0.018 mmol), Pd(OH)₂ (11 mg), dioxane (1.2 ml), water (0.25 ml), 50 h,(CH₂Cl₂/MeOH 2:1) gives XVIII (10 mg, 50%).

Synthesis of intermediate XIX: According to procedure C: XV (22 mg,0.015 mmol), Pd(OH)₂ (20 mg), dioxane (1 ml), water (0.25 ml), 22 h,(CH₂Cl₂/MeOH 15:1) gives compound XIX (13 mg, 82%). [α]_(D)−9.50°(c=1.21, MeOH).

Synthesis of intermediate XXI: According to procedure C: XIII (21 mg,0.015 mmol), Pd(OH)₂ (20 mg), dioxane (1 ml), water (0.25 ml), 22 h,(CH₂Cl₂/MeOH 15:1) gives compound XXI (13 mg, 86%).

Synthesis of intermediate XX: A mixture of XVI (20 mg, 0.0141 mmol), 10%Pd/C (20 mg) MeOH (2 ml) and AcOH (50 μl) is stirred under a hydrogenatmosphere. When TLC control indicated completion of the reaction (after22 h), the mixture is filtered over celite and evaporated to dryness.Chromatography of the residue on silica gel (CH₂Cl₂/MeOH 2:1) gives XX(15 mg, quant.). [α]_(D)−29.32° (c=1.105, MeOH).

General Procedure D

Synthesis of product XXII: A solution of XVII (27 mg, 0.0278 mmol) indry MeOH (1 ml) and a catalytic amount of freshly prepared NaOMe isstirred at r.t. under argon. After 1 h, TLC control shows completion ofthe reaction. The reaction mixture is neutralized with powderedamberlyst-15, filtered over Celite and the filtrate evaporated todryness. The residue is dissolved in MeOH (20 ml) and filtered overion-exchange Dowex Na^(⊕). The filtrate is evaporated to dryness andchromatography (CH₂Cl₂/MeOH/water 10:4:0.8) of the crude product givesXXII (12 mg, 56%). [α]_(D)−97.49° (c=0.546, MeOH).

Synthesis of product XXIII: According to procedure D: XVIII (9.0 mg,0.0083 mmol), 2 h, (CH₂Cl₂/MeOH/water 10:3:0.5) gives compound XXIII(7.5 mg, quant.). [α]_(D)−79.50° (c=0.400, MeOH).

Synthesis of product XXIV: According to procedure D: XIX (23 mg, 0.0212mmol), MeOH (2 ml), 2.5 h, (CH₂Cl₂/MeOH/water 10:3:0.5) gives compoundXXIV (7.0 mg, 39%). [α]_(D)−54.54° (c=0.586, MeOH).

Synthesis of product XXV: According to procedure D: XX (19 mg, 0.0180mmol), MeOH (2 ml), 4 h, (CH₂Cl₂/MeOH/water 10:3:0.5) gives compound XXV(10.5 mg, 70%.) [α]_(D)−69.96° (c=0.793, MeOH).

Synthesis of product XXVI: According to procedure D: XXI (19 mg, 0.0180mmol), MeOH (2 ml), 4 h, (CH₂Cl₂/MeOH/water 10:3:0.5) gives compound)XXVI (10 mg, 65%.).

Example 21 Assay for E-Selectin Antagonist Activity (FIG. 17A)

Wells of a microtiter plate (plate 1) are coated with E-selectin/hlgchimera (GlycoTech Corp., Rockville, Md.) by incubation for 2 hr at 37°C. After washing the plate 5 times with 50 mM TrisHCl, 150 mM NaCl, 2 mMCaCl₂, pH 7.4 (Tris-Ca), 100 μl of 1% BSA in Tris-Ca/Stabilcoat(SurModics, Eden Prairie, Minn.) (1:1, v/v) are added to each well toblock non-specific binding. Test compounds are serially diluted in asecond low-binding, round bottomed plate (plate 2) in Tris-Ca (60μl/well). Preformed conjugates of SLea-PAA-biotin (GlycoTech Corp.,Rockville, Md.) mixed with Streptavidin-HRP (Sigma, St. Louis, Mo.) areadded to each well of plate 2 (60 μl/well of 1 μg/ml). Plate 1 is washedseveral times with Tris-Ca and 100 μl/well are transferred from plate 2to plate 1. After incubation at room temperature for exactly 2 hours theplate is washed and 100 μl/well of TMB reagent (KPL labs, Gaithersburg,Md.) is added to each well. After incubation for 3 minutes at roomtemperature, the reaction is stopped by adding 100 μl/well of 1 M H₃PO₄and the absorbance of light at 450 nm is determined by a microtiterplate reader.

Example 22 Assay for P-Selectin Antagonist Activity (FIG. 17B)

The neoglycoprotein, sialylLe^(a)-HSA (Isosep AB, Sweden) is coated ontowells of a microtiter plate (plate 1) and the wells are then blocked bythe addition of 2% bovine serum albumin (BSA) diluted in Dulbecco'sphosphate-buffered saline (DPBS). In a second microtiter plate (plate2), test antagonists are serially diluted in 1% BSA in DPBS. Afterblocking, plate 1 is washed and the contents of plate 2 are transferredto plate 1. Pselectin/hlg recombinant chimeric protein (GlycoTech Corp.,Rockville, Md.) is further added to each well in plate 1 and the bindingprocess is allowed to incubate for 2 hours at room temperature. Plate 1is then washed with DPBS and peroxidase-labelled goat anti-human Ig(γ)(KPL Labs, Gaithersburg, Md.) at 1 μg/ml is added to each well. Afterincubation at room temperature for 1 hour, the plate is washed with DBPSand then TMB substrate (KPL Labs) is added to each well. After 5minutes, the reaction is stopped by the addition of 1 M H₃PO₄.Absorbance of light at 450 nm is then determined using a microtiterplate reader.

Example 23 Anti-Inflammatory Mouse Model. Effects of Test Compound onIL-1β-Induced Neutrophil Migration to an Air Pouch In Vivo. (FIG. 19)Animals.

Male outbred Swiss albino mice (15-18 g body weight) are purchased fromBantin and Kingman (T. O. strain; Hull, Humberside) and maintained on astandard chow pellet diet with tap water ad libitum and a 12:00 hlight/dark cycle. All animals are housed for 7 days prior toexperimentation to allow body weight to Reach ˜25 g on the day of theexperiment (day 6; see below)

Experimental Design.

Air-pouches are formed on the back of mice by air injection (2.5 mls.c.) on day 0 and day 3 (Perretti & Flower, 1993). A homogenoussuspension of carboxymethylcellulose (CMC) is made at 0.5% w/v in PBSand murine recombinant IL-1β added to it at a concentration of 20 ng/ml.

Test Compound is given at time 0 just before IL-1β administration. Anextra group is also added, in which a group of mice received CMC only(no IL-1β) to provide a basal negative control.

In all cases, air-pouches are washed 4 h after IL-1β with 2 ml of PBScontaining 3 mM EDTA, and the number of migrated leukocytes (≧90%polymorphonuclear leukocytes, PMN) determined, by taking an aliquot (100μl) of the lavage fluid and diluting 1:10 in Turk's solution (0.01%crystal violet in 3% acetic acid). The samples are then vortexed and 10μl of the stained cell solution are placed in a Neubauerhaematocymometer and neutrophils numbers counted using a lightmicroscope (Olympus B061).

Compound Administration.

On the day of the experiment, a fresh solutions of Test Compound isprepared in PBS Dulbecco's buffer supplemented with 1 mM CaCl₂ andMgCl₂. Monoclonal antibodies (mAb) against mouse P- or E-selectin arepurchased from BD Pharmingen, whereas the anti-L-selectin mAb is fromSerotec:

Rat anti-mouse L selectin (clone MEL-14):   1 mg Rat anti-mouseP-selectin (clone RB40.34): 0.5 mg/ml Rat anti-mouse E-selectin (clone10E9.6): 0.5 mg/ml

FIG. 19.

Mouse 6-day old air-pouches are inflamed with IL-1β (10 ng) at time 0;Example 17 glycomimetic-BASA (FIG. 13) and Example 12 glycomimetic-BASA(FIG. 8C) are given I.v. at time 0 and 4 h; and the mix of anti-selectinmAb is given I.v. at time 0.

Air-pouches are washed at the 8 h time-point and the number of migratedPMN determined by staining and light microscopy.

The n number is 9, 8, 7 and 8 mice per group A, B, C and D,respectively, in FIG. 19.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A compound or physiologically acceptable salt thereof, having theformula:

wherein:

where n=0-2, and R⁸ are independently selected where n=2; R²═H, —C(═O)OXwhere X is C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or C₁-C₁₄ aryl,—C(═O)NH(CH₂)_(n)NH₂, —[C(═O)NH(CH₂)_(n)NHC(═O)]_(m)(L)_(m),Z, wheren=0-30, m=0-1, Lisa linker, and Z is a benzyl amino sulfonic acid, abenzyl amino carboxylic acid, a polyethylene glycol, or a secondcompound or salt thereof having the above formula to form a dimer whereR² of the second compound or salt thereof has m=0, no Z, and is thepoint of attachment; R³═—OH,

—O—C(═O)—X, —NH₂, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2 and X isindependently selected from C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,

and any of the above ring compounds may be substituted with one to threeindependently selected of Cl, F, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈alkynyl, C₁-C₁₄ aryl, or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl,C₁-C₈ alkynyl, or C₁-C₁₄ aryl;

6′sulfated GlcNAc, 6′carboxylated GlcNAc, 6′sulfated GalNAc, 6′sulfatedgalactose, 6′carboxylated galactose or

where R⁹ is aryl, heteroaryl, cyclohexane, t-butane, adamantane, ortriazole, and any of R⁹ may be substituted with one to threeindependently selected of Cl, F, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈alkynyl or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynylor C₁-C₁₄ aryl; R⁵═H, or R⁴ and R⁵ are taken together to form

where R¹⁰ is aryl, heteroaryl,

where n=0-10, and any one of the above ring compounds may be substitutedwith one to three independently selected of Cl, F, C₁-C₈ alkanyl, C₁-C₈alkenyl, C₁-C₈ alkynyl or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenylor C₁-C₈ alkynyl; R⁶═H, fucose, mannose, arabinose, galactose orpolyols; R⁷═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or

R⁸═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,

where n=0-3 and X is independently selected from H, OH, Cl, F, N₃, NH₂,C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, C₁-C₁₄ aryl, OC₁-C₈alkanyl, OC₁-C₈ alkenyl, OC₁-C₈ alkynyl, and OC₁-C₁₄ aryl, and any ofthe above ring compounds may be substituted with one to threeindependently selected of Cl, F, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈alkynyl, C₁-C₁₄ aryl or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl,C₁-C₈ alkynyl, or C₁-C₁₄ aryl.
 2. The compound or salt thereof accordingto claim 1 wherein R⁶ is fucose.
 3. The compound or salt thereofaccording to claim 1 wherein R⁷ is H.
 4. The compound or salt thereofaccording to claim 1 wherein R⁴ is


5. The compound or salt thereof according to claim 1 wherein R⁴ is

where R⁹ is defined according to claim
 1. 6. The compound or saltthereof according to claim 5, where R⁹ is cyclohexane.
 7. The compoundor salt thereof according to claim 1 wherein R⁶ is galactose.
 8. Thecompound or salt thereof according to claim 1 wherein R⁸ is


9. The compound or salt thereof according to claim 1 wherein R² is—[C(═O)NH(CH₂)_(n)NHC(═O)]_(m)(L)_(m)Z, where n, m, L and Z are definedaccording to claim
 1. 10. The compound or salt thereof according toclaim 9 wherein Z is a benzyl amino sulfonic acid, a benzyl aminocarboxylic acid or a polyethylene glycol.
 11. The compound or saltthereof according to claim 1 wherein R³ is —O—C(═O)—X or —NH—C(═O)—X,where X is defined according to claim
 1. 12. The compound or saltthereof according to claim 11 wherein X is


13. The compound or salt thereof according to claim 1 wherein R⁵ is H.14. The compound or salt thereof according to any one of claims 1-13wherein L is a polyethylene glycol or a thiadiazole.
 15. A compositioncomprising a compound or salt thereof according to claim 1 incombination with a pharmaceutically acceptable carrier or diluent.
 16. Acompound or physiologically acceptable salt thereof comprising acompound or salt thereof according to claim 1 attached to a diagnosticor therapeutic agent.
 17. A composition comprising a compound or saltthereof according to claim 16 in combination with a pharmaceuticallyacceptable carrier or diluent.